Transporters and ion channels are integral membrane proteins vital for the traffic of ions through cell membranes. This traffic is essential for a variety of processes like cell volume regulation, maintenance of the membrane potential, and the generation and propagation of electrical signals throughout biological systems. In short, these proteins keep cells alive by finely tuning ion fluxes. My main objective is to understand the mechanisms by which transporters and ion channels regulate the flow of ions through their permeation pathways. Our approach is to study the subtle functional properties of these proteins in great detail, with the expectation that they will yield insights into the fundamental mechanisms of ion transport. PUMPS. We use the Na/K pump as a model for our study on ion pumps. The Na/K pump is a P-type ATPase present in almost all animal cells. In each pump cycle, the protein exports 3 Na+ ions and imports 2 K+ ions, at the expense of one molecule of ATP, and it goes through this cycle at a rate of ~100 times per second. Clinically, the Na/K pump is important because it is the receptor of digoxin, a widely prescribed cardiac steroid used to control some cardiac arrhythmias. With the Na/K pump we would like to understand how ions move through the protein and what are the protein conformational changes that allow the ions to access their pathways. For these studies we use the classical squid giant axon membrane preparation, which allows us access to both sides of the membrane, as well as ideal voltage-clamp conditions. In the past five years, we have learned a lot about the steps involving the movement of Na+ ions through an unperturbed pump. Now, we are using an approach fairly common among biophysicists: perturb the system and learn its behavior. To perturb the Na/K pump, we use palytoxin, a marine toxin from a coelenterate of the genus Palythoa, which somehow alters the pump cycle and allows the Na/K pump to permeate ions at very high rates, resembling more the behavior of an ion channel. Now that we have the pump working as a channel, we can ask similar questions about permeation and gating, as an ion channel biophysicist do with ion channels. Understanding the fundamental permeation properties in the palytoxin modified pumps will set the grounds for our future attempt to use this model to investigate where and how ions move through the unmodified Na/K pump. Another long-term project in the lab arises from a simple general biological question: How could an animal survive in the Antarctica with water temperatures below 0?C? In particular, how does an enzyme, like the Na/K pump, manage to function when it is subjected to below-freezing temperatures? The Na/K pump has a turnover rate of ~100 per second at room temperature. Since the transport process has a high temperature dependent coefficient (Q10 ~ 4), it is expected that at 0 degree, the pump should be pumping at about 2.5 cycles per second at most. At these low turnover rates, the Na/K pumps would not be able to keep up with the Na+ leak, a process much less sensitive to temperature. We could think of at least three plausible possibilities: 1) The cell membranes are tighter at 0 degree, so there is much less Na+ leak and consequently pumps will not be required to work at 100 cycles per second; 2) the density of pumps is higher or 3) somehow the pumps are designed to work at 100 cycles per second at 0 degree. Answering these questions is not a trivial task. For example, testing the first two possibilities will require a visit to the scientific station in Antarctica. However, we can make some progress (to justify a trip to Antarctica) on the third possibility. We are presently cloning the Na/K pumps from two closely related species of octopus, one that lives in the Antarctica and the other from the tropical waters of the Pacific Ocean. If pumps from the Antarctic octopus are pumping at rates comparable than those of the Tropical octopus, it is likely that we will find amino acid substitutions at critical positions in the protein that will lower the energetic barriers for the transport cycle. Once we obtain the full sequences, we will proceed with functional studies to characterize the biophysical properties of these closely related pumps. A question that derives from this problem relates to the mechanisms by which an individual adapts to a drastic change in temperature within its lifetime. For example, some squids migrate from waters that differ more than 10 degrees, and judging by looking at them, they do not seem to be numbed in chilling waters. We hypothesize that RNA editing, a post-transcriptional modification, is a major mechanism in adaptation. RNA editing is the enzymatic conversion of adenosine to inosine, which can be tracked as a variation from adenosine to guanine between the genomic and the cDNA sequences. This conversion provides a vast mutagenic repertoire that can be an effective mechanism to alter critical positions in a protein. We have cloned the Na/K pump from the squid Loligo opalescens and we have found many potential RNA editing sites. We are in the process of confirming with the genomic sequence and we will proceed with experiments to determine the functional consequences of these alterations. This prject is beeing initiated with our extramural collaborator Dr. Joshua Rosenthal CHANNELS. We use cyclic nucleotide-gated (CNG) Channels as model for our study on ion channels. The CNG channels are key components in the transduction of visual and olfactory signals. The role of CNG channels is to respond to changes in the intracellular concentration of cyclic nucleotides, which converts the stimuli into an electrical signal that set the levels of neurotransmitter release at the first synapse of these sensory systems. Mutations in these proteins are the causes of some diseases like retinitis pigmentosa and achromatopsia. With a long-term perspective, our purpose is to understand the molecular events underlying the activation of CNG channels by cyclic nucleotide. In other words, what are the mechanisms by which CNG channels open and close. Understanding the details of this process will teach us general principles underlying the coupling between agonist binding and allosteric transitions. We are approaching this problem both, functionally and structurally. Functionally, we are testing the role of regions believed to be involved in gating following the standard molecular biology techniques combined with functional assays. Structurally, we are embarking in a broad project to produce, purify, reconstitute and crystallize a bacterial channel that contains a cyclic nucleotide binding domain. We are in the process of assessing if we will be able to produce protein in concentrations that will allow us to pursue functional and structural studies with this bacterial channel. If we succeed, we will be able to reconstitute the channel in bilayers to perform functional studies and to begin crystallization attempts for subsequent structural studies. With a more short-term perspective, we ask questions directed toward understanding general principles about the interactions between ions and membrane channels. What are the interactions between permeant ions and the gating machinery? How different ions interfere with the gating process? How many ions a pore can accommodate at a certain time? In addition, following a more traditional pharmacological approach, we are also interested in understanding the interactions between exogenous molecules and CNG channels, which provide useful structural and functional information.